Chimeric genes can be transferred to airway epithelial cells in vitro and in vivo by a protein and DNA complex which targets the polymeric immunoglobulin receptor (plgR). This complex consists of the Fab portion of antibody against the plgR, covalently linked to poly-L-lysine, noncovalently condensed with plasmid DNA. Intravenous injection of this complex in rats results in expression of the reporter genes at high levels in the lung for 12 days, and reporter genes are detected in 10-75% of airway epithelial cells and tracheal glands. Rare hepatocytes also express reporter genes, but tissues which do not express the plgR do not. Complex prepared with irrelevant Fab fragments is ineffective. Thus, this complex provides specific targeting of foreign genes to plgR-bearing cells with reasonable efficiency and duration of expression. The cells targeted by the plgR include appropriate targets for gene therapy of cystic fibrosis (CF). The basic cell physiology of this gene transfer system must be elucidated if its properties are to be improved. This proposal is aimed at determining the intracellular fate of the gene transfer complex in plgR-bearing cells in culture. Using fluorescence and electron microscopy we will analyze the trafficking of the complex. Reagents known to affect the trafficking the native plgR with or without its natural ligand, dlgA, will be used to probe the trafficking of the gene transfer complex. With greater understanding of the intracellular processing of the complex, we may be able to enhance and prolong transgene expression. A second aim of this proposal is to explore the chemistry of the complex in the hope of enhancing gene uptake and expression. Improvements in the antibody portion, the nature and length of the polycation linker, and the plasmid DNA will be tested, including strategies to improve the persistence of DNA in the transfected cells. A third aim of the proposal is to discover alternative receptors for receptor-mediated gene transfer into cells relevant to gene therapy of CF. Binding proteins of respiratory viruses will be tested as potential ligands for the gene transfer complex which can target the apical surface of the cell. Transferrin will be assessed as a ligand, with genes coupled to the SP-C or other lung-specific promoters, to approach the small airways from the submucosa. Successful completion of the studies in this proposal will allow improvement of the duration and intensity of expression of transgenes delivered by receptor-mediated means and allow refinement of the strategy of receptor-mediated gene transfer for gene therapy of cystic fibrosis. Receptor-mediated gene transfer deserves pursuit as a potential means of human gene therapy because it is exquisitely specific, efficient, nontoxic, and can target the submucosal gland cells as well as the airway epithelium.